This is a continuation-in-part of a co-pending U.S. application having serial No. 07/539,763, filed on Jun. 18, 1990 and entitled xe2x80x9cIncreased Starch Content in Plantsxe2x80x9d.
Recent advances in genetic engineering have provided the requisite tools to transform plants to contain foreign genes. It is now possible to produce plants which have unique characteristics of agronomic and crop processing importance. Certainly, one such advantageous trait is enhanced starch content and quality in various crop plants.
Starch is a polysaccharide primarily composed of glucose units connected by alpha 1-4 and alpha 1-6 linkages. It is found in plant cells as water-insoluble grains or granules. During photosynthesis, starch is produced and stored in chloroplasts. Starch is also synthesized in roots and storage organs such as tubers and seeds. In these non-photosynthetic tissues, the starch is found in a form of plastids called amyloplasts. As in the chloroplasts, starch is stored in the amyloplasts as starch granules. The size of the granules varies depending on the plant species.
Starch is actually composed of amylose and amylopectin, two distinct types of glucose polymers. Amylose is composed primarily of linear chains of alpha 1-4 linked glucose molecules. On average, amylose has a chain length of about 1000 glucose molecules. Amylopectin contains shorter chains linked together with alpha 1-6 linkages. On average, amylopectin has a chain length of about 20-25 glucose molecules.
Until recently, there was controversy in the literature as to whether ADPglucose or UDPglucose was the substrate for starch synthesis. With the isolation of Arabidopsis mutants lacking ADPglucose pyrophosphorylase it is now accepted that plants use ADPglucose as the substrate for starch synthesis. There are three steps in the synthesis of starch. All these reactions take place within the chloroplasts or amyloplasts. In the first step, ADPglucose is produced from glucose-1-phosphate and ATP by ADPglucose pyrophosphorylase (EC 2.7.7.27). In the second step, ADPglucose is used by starch synthase (EC 2.4.1.21) to form linear chains of starch containing the xcex1, 1-4 linkage. In the third step, the branching enzyme(s) (EC 2.4.1.18) introduce alpha 1-6 linkages to produce the amylopectin molecule.
The controlling step in the synthesis of starch in plants has been a topic of dispute. Although synthesis of ADPglucose by ADPglucose pyrophosphorylase has been proposed to be the controlling step in starch biosynthesis, this has not been proved. In fact, European Patent Application publication number 0368506 A2, which concerns ADPglucose pyrophosphorylase, questions the role of the enzyme as the rate limiting step in starch biosynthesis. An argument against ADPglucose pyrophosphorylase being the controlling enzyme can be made from the results with an Arabidopsis mutant (Lin, 1988a,b). This mutant, TL46, was found to contain only about 5% of the ADPglucose pyrophosphorylase activity compared to the wild type plants. However, TL46 plants still produced about 40% of the wild type starch levels. If ADPglucose pyrophosphorylase is the rate limiting enzyme, one would have expected a 95% reduction in enzyme activity to produce more than a 60% reduction in starch accumulation. Similarly, the in vitro measurements on extractable activities suggest this enzyme can only be rate limiting if its in vivo activity is substantially inhibited by the allosteric regulators of the enzyme activity.
The present invention provides structural DNA constructs which encode an ADPglucose pyrophosphorylase (ADPGPP) enzyme and which are useful in producing enhanced starch content in plants. It is also demonstrated that the ADPGPP enzyme activity in plant cells and tissues is a controlling step in starch biosynthesis.
In accomplishing the foregoing, there is provided, in accordance with one aspect of the present invention, a method of producing genetically transformed plants which have elevated starch content, comprising the steps of:
(a) inserting into the genome of a plant cell a recombinant, double-stranded DNA molecule comprising
(i) a promoter which functions in plants to cause the production of an RNA sequence in target plant tissues,
(ii) a structural DNA sequence that causes the production of an RNA sequence which encodes a fusion polypeptide comprising an amino-terminal plastid transit peptide and an ADPglucose pyrophosphorylase enzyme,
(iii) a 3xe2x80x2 non-translated DNA sequence which functions in plant cells to cause transcriptional termination and the addition of polyadenylated nucleotides to the 3xe2x80x2 end of the RNA sequence;
(b) obtaining transformed plant cells; and
(c) regenerating from the transformed plant cells genetically transformed plants which have an elevated starch content.
In accordance with another aspect of the present invention, there is provided a recombinant, double-stranded DNA molecule comprising in sequence:
(a) a promoter which functions in plants to cause the production of an RNA sequence in target plant tissues;
(b) a structural DNA sequence that causes the production of an RNA sequence which encodes a fusion polypeptide comprising an amino-terminal plastid transit peptide and an ADPglucose pyrophosphorylase enzyme; and
(c) a 3xe2x80x2 non-translated region which functions in plant cells to cause transcriptional termination and the addition of polyadenylated nucleo-tides to the 3xe2x80x2 end of the RNA sequence, said promoter being heterologous with respect to the structural DNA.
There has also been provided, in accordance with another aspect of the present invention, bacterial and transformed plant cells that contain, respectively, DNA comprised of the above-mentioned elements (a), (b) and (c).
In accordance with yet another aspect of the present invention, differentiated plants are provided that have increased starch content.